Demodex (folliculorum and brevis) mites are microscopic ectoparasites that commonly infest the pilosebaceous unit of the skin. Uncontrolled overgrowth of these mites has been implicated in causing rosacea in the face, blepharitis in the eyelid, and other inflammation-associated conjunctival and corneal abnormalities. We have conducted a number of studies to substantiate the potential pathogenicity of demodex mites in causing eye irritation and inflammation in patients. We have improved the accuracy of diagnosing demodex infestation based on sampling lashes from the patient, and established that lashes harboring cylindrical dandruff are a reliable sign indicative of demodex infestation. Furthermore, we have developed an in vitro assay to screen agents that can potentially kill adult demodex mites. Using this assay, we have discovered that the Baby Shampoo does not, but tea tree oil (TTO), an essential oil distilled from Melaleuca alternifolia does, kill mites. We further showed that weekly office lid scrub with 50% TTO solution followed by daily home lid hygiene with tea tree shampoo can eradicate ocular Demodex in a majority of the suffering patients. However, this regimen achieves eradication of demodex infestation in the majority, but not all, of the patients, generates eye irritation in some patients, and is still not simple enough for patients to self administer it at home. In the SBIR Phase I application, we would like to identify the most active ingredient in TTO that may kill adult demodex mites in vitro, to examine the safety/toxicity of ophthalmic formulations containing this active ingredient in an in vitro cytotoxicity test and rabbit eyes, and to determine the stability of such formulations under different storage conditions and durations. Accomplishment of these three aims will allow us to collect sufficient data for obtaining an IND approval from the FDA so that we may conduct, in Phase II, clinical studies, eventually leading to the development of the first FDA-approved prescription ophthalmic formulation containing the most active ingredient in TTO to eradicate ocular demodex infestation in this most common eye disease. PUBLIC HEALTH RELEVANCE: This research is aimed to developing the first FDA-approved prescription drug containing the most active ingredient in tea tree oil to eradicate Demodicosis (the microscopic infestation of the hair follicle and the oil gland by Demodex mites) commonly occurring in the skin and the eyelid. Demodicosis is one of the leading causes of blepharitis, a common eye disease, for which eradication has not been achieved by any over-the-counter lid scrub regimen. Because Demodex infestation is prevalent in the older population, an effective treatment as such for eradicating Demodicosis for the eye will address an important public health concern when the aging population is increased, and help to develop a similar treatment for the skin in the future.